Human babesiosis is a malaria-like multisystem disease caused primarily by an emerging apicomplexan parasite Babesia microti that infects and develops within human erythrocytes. The parasite is transmitted to humans by the tick vector, Ixodes scapularis and can also be introduced through blood transfusion. Babesiosis treatment options consist of two combinations of antimalarials, azithromycin+atovaquone or clindamycin+quinine. However, even with these drugs and other supportive measures such as exchange transfusion, the disease can be fatal in elderly individuals or immunocompromised patients. In order to develop better therapies and diagnostic tests for human babesiosis, we developed a short-term ex vivo proliferation assay to test the efficacy of the four currently clinically used drugs as well as compounds known to have activity against other protozoa. Our studies demonstrated potent activity of endochin-like quinolones (ELQs) and atovaquone against B. microti and indicated limited activity of other drugs used in babesiosis therapy. In vivo efficacy studies identified 2 compounds ELQ-271 and ELQ-316 with potent activity against the B. microti LabS1 strain following oral administration at 10mg/kg. However, we found that monotherapy involving either ELQs or atovaquone was always accompanied by recrudescence 9 to 16 days post-drug removal, emphasizing the need to identify better analogs of these compounds or a suitable therapeutic strategy that can lead to radical cure. Remarkably, we found that a combination therapy consisting of atovaquone and a prodrug of ELQ-316, ELQ-334, results in radical cure of experimental babesiosis in mice at doses as low as 2.5+2.5 mg/kg. Biochemical assays demonstrated inhibition of the activity of the B. microti cytochrome bc1 complex by ELQ, and analysis of parasites resistant to either ELQs or atovaquone identified single mutations in the B. microti Cytochrome b (BmCytb) gene. Building upon these data, we propose the following three specific aims. In Aim 1, we will characterize the activity and selectivity of ELQ-316 and 30 structurally diverse ELQs. Biochemical assays will be used to characterize their activity against the B. microti and human bc1 complex, and cell culture assays will be used to assess their efficacy against the parasite and evaluate their toxicity on mammalian cells. Compounds with the greatest therapeutic indices will be further examined for their potency in mice and ability to clear infection without recrudescence. In Aim 2, we will synthesize prodrugs of the 2 most potent ELQs selected from aim 1. The ELQ-prodrug strategy that yields the greatest oral bioavailability will be tested in combination with atovaquone in comparison to the ELQ-334+Atovaquone combination. In Aim 3, we will perform biochemical and genetic assays to elucidate the mechanism of action of ELQs, identify possible routes for drug failure and design the best therapeutic strategy for an ideal formulation that achieves radical cure. The success of the proposed studies will set the stage for future clinical trials to create a treatment regimen for human babesiosis that is safe and achieves radical cure.